WO2023109881A1 - High-efficiency and low-toxicity dna and rna lipid delivery carrier - Google Patents

Abstract

Provided in the present invention is a high-efficiency and low-toxicity DNA and RNA lipid delivery carrier. In order to enrich the types of nucleic acid drug delivery carriers, improve the in-vivo delivery and expression effects of the nucleic acid drug and reduce the toxicity and side effects, a new ionizable lipid compound having a special structure is provided in the present invention. The compound has a good binding effect with a negatively-charged nucleic acid, which effectively prevents the premature degradation of the nucleic acid by a nuclease in a cell and facilitates the passage of a liposome loaded with the nucleic acid through a cell membrane. Effective degradation and rapid removal can be achieved, the toxicity and side effects of the nucleic acid drug are reduced, and a nucleic acid drug delivery carrier truly having a high transfection efficiency, a high expression level and a low toxicity can be formed.

Description

A high-efficiency and low-toxicity DNA and RNA lipid delivery carrier technical field

The invention belongs to the technical field of nucleic acid drug delivery carrier, and in particular relates to a high-efficiency and low-toxicity DNA and RNA lipid delivery carrier.

Background technique

At present, lipid delivery vehicles have been widely used for drug delivery, especially with the development of nucleic acid drugs, the application of lipid delivery vehicles has expanded rapidly. Since the nucleic acid molecule itself has negative charge, it is not conducive to the function of the cell membrane, and the cell permeability is poor, and the nucleic acid in the naked state entering the tissue or cell is easily degraded by nucleases. Nucleic acid transfection not only requires the nucleic acid with biological functions To be successfully transported into the cell, it is also necessary to ensure that the nucleic acid can maintain its biological function in the cell. Carriers commonly used in research and clinical applications include viral vectors and non-viral vectors. Liposome vectors are non-viral vectors. Compared with viral vectors, they have higher nucleic acid transfection efficiency, lower cytotoxicity, and more convenient preparation process. It has higher practicality, so it has received more and more attention.

With the deepening of research, relevant researchers have actively developed a variety of liposome carriers to continuously improve safety and efficiency. The carrier formed by the most common cationic lipid compound in the early stage is: a liposome-nucleic acid drug complex is formed by a positively charged cationic lipid compound and a negatively charged nucleic acid, and the cationic liposome-nucleic acid drug complex The entire surface of the object is positively charged, adsorbed to the negatively charged cell surface through electrostatic interaction, enters the cell through endocytosis, and forms endosomes. The cationic lipid in the cationic liposome interacts electrostatically with the negatively charged lipid in the endosome, and the negatively charged lipid flips from the lumen of the endosome to the lumen, forming neutral ions with the positively charged lipid Yes, nucleic acid drugs are separated from cationic lipids. In 2018, the FDA approved the first siRNA drug (patisiran [Onpattro]), which uses Dlin-MC3-DMA liposome as a delivery vehicle. Recently, ionizable lipids have attracted attention due to their ability to change their electrical charge in response to the pH in the environment.

Although ionizable lipids have made recent progress in drug delivery, and have shown obvious advantages over viral vectors and other types of non-viral vectors in terms of encapsulation efficiency, nucleic acid expression, and cytotoxicity, they are currently available for use. There are still very few ionizable lipid molecules, and most of them are easily distributed to the liver, which increases the metabolic burden of the liver and produces toxic and side effects. Therefore, it is still necessary to explore more ionizable lipid compounds suitable for the application of nucleic acid drugs, and to develop nucleic acid drug delivery vehicles that truly take into account high transfection efficiency, high expression effect and low toxicity.

Contents of the invention

The purpose of the present invention is to provide an ionizable lipid compound that has a simple preparation method, is easy to combine with nucleic acid, and is easy to degrade, which enriches the types of ionizable lipid compounds and provides more options for nucleic acid drug delivery.

Another object of the present invention is to provide a delivery carrier, which is a highly efficient and low-toxic lipid delivery carrier for delivering DNA and RNA. The delivery carrier and the nucleic acid molecule encapsulated in the delivery carrier form a pharmaceutical composition, which ensures the activity of the nucleic acid drug and high expression efficiency of the nucleic acid molecule, and at the same time significantly reduces the distribution of the drug in the liver.

In order to solve the problems of the technologies described above, the present invention adopts the following technical solutions:

Ionizable lipid compounds represented by general formula (I) and general formula (II),

in,

In the general formula (I), G ₁ is a C ₁ -C ₁₀ linear alkylene group, G ₂ is -OC(=O)R, -C(=O)OR or -C(=O)NHR, and R is C ₅ -C ₂₀ straight chain alkyl or

_R1 is hydrogen, methyl, ethyl or isopropyl, m is an integer between 1 and 10, n is an integer between 1 and 5, and f is an integer between 1 and 5;

In the general formula (II), G ₃ is a C ₁ -C ₁₀ linear alkylene group, and G ₄ is -OC(=O)R', -C(=O)OR' or -C(=O)NHR' , R' is

R ₂ and R ₃ are independently hydrogen, methyl, ethyl or isopropyl, and q is an integer between 1 and 3.

Preferably, said G ₁ is a C ₂ -C ₈ linear alkylene group.

Preferably, said G ₃ is a C ₅ -C ₁₀ linear alkylene group.

Preferably, said R ₁ is hydrogen.

Preferably, said m is an integer between 3-8, more preferably an integer between 4-6.

Preferably, said f is an integer between 1 and 4, more preferably 2 or 3.

Preferably, said R is C ₅ -C ₁₅ linear alkyl or

Preferably, said G ₂ is -C(=O)OR.

Preferably, said G ₄ is -C(=O)OR'.

Preferably, one of said _R2 and said _R3 is hydrogen, and the other is methyl, ethyl or isopropyl.

Further preferably, one of said R ₂ and said R ₃ is hydrogen, and the other is methyl.

Still further preferably, said R ₂ is methyl, and said R ₃ is hydrogen.

According to some specific and preferred embodiments, the ionizable lipid compound is one or more of the following compounds:

The present invention also provides a delivery carrier, which includes one or more of the ionizable lipid compounds represented by the general formula (I) and the general formula (II).

Preferably, the delivery vehicle further includes auxiliary molecules.

Further preferably, the molar ratio of the ionizable lipid compound and the auxiliary molecule is (0.1-1): (0.1-1), more preferably (0.5-1): (0.5-1 ).

The auxiliary molecule may be an auxiliary molecule commonly used in the art.

Preferably, the auxiliary molecules include artificially synthesized or naturally derived auxiliary lipids or lipid molecules, any species of animal sources, and any types of cells or vesicles (including exosomes) or their components, One or more of polypeptide molecules, polymer molecules, sugar molecules or inorganic substances.

Further preferably, the auxiliary molecules include cholesterol, calcipotriol, stigmasterol, β-sitosterol, lupeol, betulin, ursolic acid, oleanolic acid, dioleoylphosphatidylcholine , Distearoylphosphatidylcholine, 1-stearyl-2-oleoyl lecithin, dioleoylphosphatidylethanolamine, (1,2-dioleoxypropyl) trimethylammonium chloride, bis Decyldimethylammonium bromide, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 5000, distearyl One or more of acylphosphatidylethanolamine-polyethylene glycol 2000, activated carbon, silicon dioxide, and calcium phosphate.

Preferably, the ionizable lipid compound and/or the auxiliary molecule is modified with a targeting substance.

Still further preferably, the targeting substance includes one or more of folic acid, single-chain antibody or targeting polypeptide.

Preferably, the delivery carrier is nano-lipid particles.

Further preferably, the average size of the nanoparticle preparation is 50nm-200nm.

Still further preferably, the average size of the nanoparticle preparation is 50nm-150nm.

Further, the polydispersity index of the nanoparticle preparation is ≤0.4.

Still further, the polydispersity index of the nanoparticle preparation is ≤0.3.

The delivery carrier of the present invention and the nucleic acid molecule encapsulated in the delivery carrier together constitute a nucleic acid pharmaceutical composition.

Preferably, the nucleic acid molecule is one or more of pDNA, siRNA, ASO or mRNA.

Preferably, the mass ratio of the nucleic acid molecule to the delivery carrier is 1:(5-50); further 1:(5-40), and still more preferably 1:(5-30).

Preferably, the nucleic acid pharmaceutical composition includes pharmaceutically acceptable additives, and the additives include one or more of excipients, stabilizers or diluents.

Further, the additives include but not limited to sucrose, trehalose or other stabilizers.

Specifically, the added amount of the additive is 1%-20% of the total mass of the pharmaceutical composition.

Preferably, the nucleic acid pharmaceutical composition can be a freeze-dried powder or an injection, and the injection can be administered locally through a microneedle, injection or perfusion through the muscle, subcutaneous, endothelium, intratumoral, or intravenous injection. medicine.

In the present invention, the delivery vector or the nucleic acid pharmaceutical composition is used to deliver nucleic acid molecules to mammalian cells.

Preferably, said mammal is human.

The present invention also provides a method for delivering nucleic acid molecules in vivo, using the delivery carrier to deliver the nucleic acid molecules into the body of a subject.

Preferably, said subject is a mammal.

Further preferably, the subject is human.

Compared with the prior art, the present invention has the following advantages:

The invention provides a new ionizable lipid compound, which enriches the types of ionizable lipid compounds, and the delivery carrier formed by it has the advantages of high encapsulation efficiency and low toxicity, and can efficiently deliver nucleic acid drugs in vivo and Efficient expression provides more options for nucleic acid drug delivery, which is of great significance to the development and application of nucleic acid drugs.

Description of drawings

Fig. 1 is the hydrogen spectrogram of compound a;

Fig. 2 is the hydrogen spectrogram of compound b;

Fig. 3 is the hydrogen spectrogram of compound 1-1;

Fig. 4 is the hydrogen spectrogram of compound 2-1;

Fig. 5 is the hydrogen spectrogram of compound 3-1;

Fig. 6 is the hydrogen spectrogram of compound 1;

Fig. 7 is the high-resolution mass spectrogram of compound 1;

Fig. 8 is the hydrogen spectrogram of compound 2;

Fig. 9 is the high-resolution mass spectrum of compound 2;

Figure 10 is the hydrogen spectrogram of compound 3;

Figure 11 is the high resolution mass spectrum of compound 3;

Fig. 12 is the particle size distribution figure of the nano lipid particle prepared in embodiment 4;

Figure 13 is the in vivo delivery effect of Lipid-01 liposome mice in Example 5;

Figure 14 is the in vivo delivery effect of Lipid-02 liposome mice in Example 5;

Figure 15 is the in vivo delivery effect of Lipid-03 liposome in mice in Example 5.

Detailed ways

The present invention will be further described below in conjunction with embodiment. However, the present invention is not limited to the following examples. The implementation conditions adopted in the embodiments can be further adjusted according to the different requirements of specific use, and the unspecified implementation conditions are conventional conditions in this industry. The technical features involved in the various embodiments of the present invention may be combined with each other as long as they do not constitute conflicts with each other.

In order to reduce the toxic and side effects caused by the accumulation and expression of drug components in the liver, reduce the production cost of nucleic acid drug delivery vectors, and improve the delivery and expression effect of nucleic acid drugs in vivo, the inventors conducted a lot of research and experimental verification, and developed a new The ionizable lipid compound can form a nucleic acid drug delivery vehicle that truly combines high transfection efficiency, high expression effect and low toxicity.

Specifically, in the present invention, the ionizable lipid compound is a compound represented by general formula (I) and general formula (II):

in,

In the general formula (I), G ₁ is a C ₁ -C ₁₀ linear alkylene group, G ₂ is -OC(=O)R, -C(=O)OR or -C(=O)NHR, and R is C ₅ -C ₂₀ straight chain alkyl or

_R1 is hydrogen, methyl, ethyl or isopropyl, m is an integer between 1 and 10, n is an integer between 1 and 5, and f is an integer between 1 and 5;

In the general formula (II), G ₃ is a C ₁ -C ₁₀ linear alkylene group, and G ₄ is -OC(=O)R', -C(=O)OR' or -C(=O)NHR' , R' is

R ₂ and R ₃ are independently hydrogen, methyl, ethyl or isopropyl, and q is an integer between 1 and 3.

The ionizable lipid compound with a special structure can improve its binding ability to negatively charged nucleic acids, prevent nucleic acids from being degraded by nucleases prematurely in cells, and facilitate the liposomes loaded with nucleic acids to pass through the cell membrane. Effective degradation and rapid clearance in the body are achieved, and the toxic and side effects of nucleic acid drugs are reduced.

According to the present invention, the delivery carrier includes one or more of the ionizable lipid compounds represented by the general formula (I) and the general formula (II), and optionally includes auxiliary molecules.

According to the present invention, the delivery carrier is a lipid nanoparticle with a particle diameter of 50nm-200nm.

According to the present invention, the delivery vector can be used to deliver one or more of pDNA, siRNA, ASO or mRNA.

According to the present invention, the delivery carrier and the nucleic acid molecule encapsulated in the delivery carrier constitute a nucleic acid pharmaceutical composition, and the nucleic acid molecule is one or more of pDNA, siRNA, ASO or mRNA.

According to the present invention, the nucleic acid pharmaceutical composition can be a lyophilized powder or an injection, and the injection can be locally administered by microneedle, injection or perfusion through muscle, subcutaneous, endothelial, or intratumoral, or through intravenous injection. medication.

In the present invention, the delivery vector or the nucleic acid pharmaceutical composition is used to deliver nucleic acid molecules to mammalian cells, preferably the mammal is a human.

The technical solutions and technical effects of the present invention will be further described below in conjunction with specific embodiments.

In the following examples, unless otherwise specified, all are conventional methods; the experimental materials used, unless otherwise specified, were purchased from conventional biochemical reagent manufacturers.

Example 1

The synthetic route of compound 1:

Step 1: Synthesis of compound 1-1

8-Bromooctanoic acid (1.139g, 5.13mmol) and citronellol (1.599g, 10.25mmol) were dissolved in dichloromethane (60mL). After fully dissolving, EDC hydrochloride (0.98g, 5.13mmol) and DMAP ( 0.125 g, 1.03 mmol). The mixture was stirred at room temperature for 18 hours. After the reaction was completed, it was diluted with DCM (200 mL), and washed with saturated NaHCO ₃ (100 mL) and brine (100 mL). The combined organic layers were dried over anhydrous _Na2SO4 , and the solvent was removed in _vacuo to obtain a crude product, which was purified by chromatography (silica gel column, eluent was petroleum ether containing 0.5% EA (volume percent)), and The purified product was evaporated to remove the eluent to obtain light yellow oily compound (0.648 g, 35%). The hydrogen spectrum of compound 1-1 is shown in Figure 3.

1H NMR (400MHz, CDCl ₃ ) δ: 5.09(s, 1H), 4.18-4.01(m, 2H), 3.40(t, J=6.8Hz, 2H), 2.29(t, J=7.4Hz, 2H), 1.98(s,2H),1.84(dd,J=14.3,7.0Hz,2H),1.70-1.60(m,9H),1.38(d,J=37.7Hz,9H),0.89(t,J=12.9Hz ,4H).

Step 2: Synthesis of Compound 1

2-(bis(2-aminoethyl)amino)ethan-1-ol) (compound a, 0.044g, 0.30mmol, see Figure 1 for hydrogen spectrum) and 6-bromohexanoic acid 3,7-dimethyloctyl -6-enyl ester (0.398 g, 1.2 mmol) was added to the reaction flask and dissolved in THF/CH ₃ CN (1:1, 6 mL), followed by DIPEA (0.155 g, 1.20 mmol). The reactant was stirred at 63 °C for 72 h. After the reaction was cooled to room temperature, the solvent was removed in vacuo. The crude product was extracted with ethyl acetate and saturated NaHCO ₃ , the combined organic layers were dried over anhydrous Na ₂ SO ₄ , and the solvent was removed in vacuo to obtain the crude product, which was subjected to chromatography (silica gel column, eluent containing 2% Methanol (volume percent) in dichloromethane) was purified, and the purified product was evaporated to remove the eluent to obtain compound 1 (25.7 mg, yield 3.6%) as a yellow oil. The hydrogen spectrum of compound 1 is shown in Figure 6, and the mass spectrum is shown in Figure 7.

1H NMR (400MHz, CDCl ₃ ) δ5.09(s, 4H), 4.10(dd, J=12.6, 6.5Hz, 8H), 3.61(d, J=21.6Hz, 2H), 3.19(s, 1H), 3.03(s,1H),2.98-2.92(m,1H),2.82(s,4H),2.67(s,4H),2.28(t,J＝7.5Hz,8H),2.03-1.93(m,8H) ,1.74-1.50(m,49H),1.36(ddd,J=44.4,23.9,10.0Hz,39H),1.23-1.13(m,4H),0.91(d,J=6.5Hz,12H).

Example 2

Synthetic route of compound 2

Step 1: Synthesis of Compound 2-1

Add linalyl alcohol (0.267g, 1mmol) and triethylamine (0.133g, 1.3mmol) into the reaction flask in an ice-water bath, add dichloromethane (6mL), and dissolve acryloyl chloride (0.11g, 1.2mmol) in dichloromethane (2.2 mL) was slowly added dropwise into the reaction flask, and the reaction lasted for 10 minutes. The reaction was kept below 10° C., and finally the ice bath was removed, and the reaction solution was reacted at room temperature for 2 hours. Wash with saturated brine to obtain a crude product, which is purified by chromatography (silica gel column, eluent is petroleum ether containing 0.5% EA (volume percentage), and the purified product is evaporated to remove the eluent to obtain shallow Yellow oily compound 2-1 (0.173 g, yield: 50%), the hydrogen spectrum of compound 2-1 is shown in Figure 4.

1H NMR (400MHz, CDCl ₃ ) δ: 6.41 (dd, J = 17.3, 1.5Hz, 1H), 6.13 (dd, J = 17.3, 10.4Hz, 1H), 5.82 (dd, J = 10.4, 1.5Hz, 1H ),5.47-5.26(m,4H),4.16(t,J=6.7Hz,2H),2.78(t,J=6.5Hz,2H),2.06(dd,J=13.6,6.7Hz,4H),1.75 -1.60(m,2H),1.39–1.17(m,16H),0.88(dt,J＝10.4,5.3Hz,3H).

Step 2: Synthesis of Compound 2

1,3-diamino-2-propanol (compound b, 0.04504g, 0.50mmol, see Figure 2 for hydrogen spectrum) and 2-allylic acid (9Z, 12Z)-octadecadienyl ester (0.64g, 2mmol) was added into a reaction flask and dissolved in THF/CH ₃ CN (1:1, 6mL), and then DIPEA (0.155g, 1.20mmol) was added and reacted at 80°C for 48 hours. After the reaction was cooled to room temperature, the solvent was removed in vacuo to obtain a crude product, which was purified by chromatography (silica gel column, eluent was dichloromethane containing 0.3% methanol (volume percent)), The purified product was evaporated to remove the eluent to obtain compound 2 (20.58 mg, yield 3%) as light yellow oil. The hydrogen spectrum of compound 2 is shown in Figure 8, and the mass spectrum is shown in Figure 9.

1H NMR (400MHz, CDCl ₃ ) δ5.45-5.32(m, 16H), 4.09(t, J=6.8Hz, 8H), 3.78(d, J=20.7Hz, 1H), 3.51(d, J=27.2 Hz, 1H), 2.81(t, J=6.4Hz, 16H), 2.49(s, 10H), 2.09(q, J=6.8Hz, 16H), 1.69-1.60(m, 8H), 1.41-1.30(m ,66H),0.93(t,J=6.8Hz,12H).

Example 3

Synthetic route of compound 3

Step 1: Synthesis of compound 3-1

6-Bromohexanoic acid (1.0g, 5.13mmol) and undecyl alcohol (1.77g, 10.25mmol) were dissolved in dichloromethane (60mL), EDC hydrochloride (0.98g, 5.13mmol) and DMAP (0.125 g, 1.03 mmol). The mixture was stirred at room temperature for 18 hours. After the reaction was completed, it was diluted with DCM (200 mL), and washed with saturated NaHCO ₃ (100 mL) and brine (100 mL). The combined organic layers were dried over anhydrous _Na2SO4 , and the solvent was removed in _vacuo to obtain a crude product, which was purified by chromatography (silica gel column, eluent was petroleum ether containing 0.5% EA (volume percent)), and The purified product was evaporated to remove the eluent to obtain light yellow oily compound 3-1 (0.69 g, yield 38.6%). The hydrogen spectrum of compound 3-1 is shown in Figure 5.

1H NMR (400MHz, CDCl ₃ ) δ: 4.10(t, J=6.6Hz, 2H), 3.45(t, J=6.7Hz, 2H), 2.36(t, J=7.3Hz, 2H), 1.97-1.88( m,2H),1.68(tt,J=14.5,7.3Hz,4H),1.53(dd,J=15.1,7.9Hz,2H),1.33(d,J=16.9Hz,16H),0.92(t,J =6.5Hz,3H).

Step 2: Synthesis of compound 3

1,3-Diamino-2-propanol (0.027 g, 0.30 mmol) and undecyl 6-bromohexanoate (0.417 g, 1.20 mmol) were dissolved in THF/CH ₃ CN (1:1, 6 mL), Afterwards additional DIPEA (0.155 g, 1.20 mmol) was added. The reactant was stirred at 63 °C for 72 h. After the reaction was cooled to room temperature, the solvent was removed in vacuo. The crude product was extracted with ethyl acetate and saturated NaHCO ₃ , the combined organic layers were dried over anhydrous Na ₂ SO ₄ , the solvent was removed in vacuo to give the crude product, the crude product was passed through chromatography, the crude product was passed through chromatography Chromatography (silica gel column, the eluent is dichloromethane containing 1% methanol (volume percentage)) purification, and the purified product was evaporated to remove the eluent to obtain light yellow oily compound 3 (47.72mg, yield 4.1%) . The hydrogen spectrum of compound 3 is shown in Figure 10, and the mass spectrum is shown in Figure 11.

1H NMR (400MHz, CDCl ₃ ) δ5.30(s, 1H), 4.05(t, J=6.8Hz, 8H), 3.68(s, 1H), 2.47(s, 8H), 2.30(t, J=7.5 Hz,8H),1.62(dd,J=15.1,7.7Hz,16H),1.47(s,6H),1.28(d,J=16.8Hz,78H),0.88(t,J=6.8Hz,12H).

Example 4:

Prepare nanolipid particles and test their particle size and potential.

Preparation method of nano lipid particles:

(1) According to the ionizable compound: DSPC: DMG-PEG2000: cholesterol is 50:10:1.5:38.5 (molar ratio), with absolute ethanol as solvent, prepare liposome solution, control the sum of the concentration of each component to be 50mM, dissolve and mix well and store at -20°C for later use.

(2) Use 25mM sodium acetate buffer solution with a pH of about 5.2 to dissolve the mRNA, and prepare a nucleic acid preparation with a final concentration of about 0.1mg/mL.

(3) The liposome solution and the nucleic acid preparation are mixed with a two-phase volume ratio of about 4:1, and the total rate of the two-phase solution is 12mL/min, and the two-phase solution is mixed by manual vortexing to form nano-lipids Immediately after the particle solution is diluted with PBS buffer solution with pH 7.2 or sodium acetate buffer solution with pH 7.4 to dilute it 20 times in volume, use a 10KD ultrafiltration tube to concentrate, and the speed of the centrifuge should not exceed the speed of the ultrafiltration tube. The highest speed limit, after 2 to 3 times of changing the solution, the pH of the nano-liposome particle solution is changed from 5.2 to 7.2, and finally the nano-liposome particle solution is concentrated to a final concentration of about 200mM, and stored at 4°C Download the spare.

After the nanoliposome particle solution was diluted 50 times with 1×PBS, the particle size, PDI, of the nanoliposome particle was detected by Zetasizer Nano ZS (Malvern, Worcestershire, UK).

Zeta potential was determined by diluting nanoliposome particles into 15 mM PBS.

Quant-It RiboGreen RNA Quantitative Detection Kit was used to measure the encapsulation efficiency on Modulus microporous multifunctional detector.

The detection results of particle size, PDI, encapsulation efficiency and potential are shown in Table 1 and Figure 12.

Table 1

Embodiment 5: liposome animal transfection experiment in vivo:

Using mRNA expressing Luciferase fluorescent protein, prepare nano-lipid particles according to the preparation method of Example 4, wherein the amount of mRNA is 120 μg, and the total amount of ionizable liposome compound, DSPC, DMG-PEG2000 and cholesterol is 1200 μg, using 400 μL of neutral PBS buffer quickly switches the liposome environment.

The above-prepared nano-lipid particles were rapidly injected intramuscularly (IM) into the hindlimb medial muscles of 6-8 week female Babl/c mice, and 30 μg of mRNA were injected into the left and right hindlimbs respectively. At different time periods after injection, the expression of luciferase in the mice after injection was observed by a small animal imager.

The above-prepared nano-lipid particles were rapidly injected (IV) into 6-8 week old female Babl/c mice through the tail vein, and the amount of mRNA injected was 60 μg. At different time periods after injection, the expression of luciferase in the mice after injection was observed by a small animal imager.

After 4 hours, the heart, liver, spleen, lung, and kidney of the mice were subjected to fluorescence imaging.

The delivery effect of Lipid-01 liposome in mice is shown in Figure 13. The results show that the fluorescence expression can reach 7×10 ⁷ after 4 hours of intramuscular injection. It can be seen from the imaging of various organs of the mouse that the fluorescence in the organs after intramuscular injection The expression is mainly concentrated in the spleen (80%). After intravenous injection, the fluorescence is distributed in the liver (63%), spleen (31%) and lungs (6%), indicating that Lipid-01 intramuscular injection has good spleen targeting sex.

The delivery effect of Lipid-02 liposome in mice is shown in Figure 14. The results show that the fluorescence expression is about 10 ⁷ after 6 hours of intramuscular injection. It can be seen from the imaging of various organs of the mouse that the fluorescence in the organs after intravenous injection is mainly It is distributed in the liver, and the expression level of fluorescence in the liver of mice after intramuscular injection is significantly lower, indicating that Lipid-02 is more suitable for intramuscular injection.

The in vivo delivery effect of Lipid-03 liposome in mice is shown in Figure 15. The results show that the fluorescence expression is about 10 ⁷ 4 hours after intramuscular injection. It can be seen from the imaging of various organs of the mouse that after intramuscular and intravenous injection The fluorescence was concentrated in the spleen, indicating that both intramuscular and intravenous injections of Lipid-03 have good spleen targeting.

The present invention has been described in detail above, and its purpose is to allow those familiar with this field to understand the content of the present invention and implement it, and can not limit the protection scope of the present invention with this. Effect changes or modifications should be covered within the protection scope of the present invention.

Claims (16)

1. Ionizable lipid compounds represented by general formula (I) and general formula (II),

   

   in,

   In the general formula (I), G ₁ is a C ₁ -C ₁₀ linear alkylene group, G ₂ is -OC(=O)R, -C(=O)OR or -C(=O)NHR, and R is C ₅ -C ₂₀ straight chain alkyl or

   

   _R1 is hydrogen, methyl, ethyl or isopropyl, m is an integer between 1 and 10, n is an integer between 1 and 5, and f is an integer between 1 and 5;

   In the general formula (II), G ₃ is a C ₁ -C ₁₀ linear alkylene group, and G ₄ is -OC(=O)R', -C(=O)OR' or -C(=O)NHR' , R' is

   

   R ₂ and R ₃ are independently hydrogen, methyl, ethyl or isopropyl, and q is an integer between 1 and 3.
2. The ionizable lipid compound according to claim 1, wherein said _G is a C ₂ -C ₈ linear alkylene group;

   And/or, said G ₃ is a C ₅ -C ₁₀ linear alkylene group;

   And/or, said R ₁ is hydrogen;

   And/or, said m is an integer between 3 and 8,

   And/or, said f is an integer between 1 and 4;

   And/or, said R is C ₅ -C ₁₅ linear alkyl or

   
3. The ionizable lipid compound according to claim 2, wherein said m is an integer between 4 and 6;

   And/or, said f is 2 or 3. 4. The ionizable lipid compound according to claim 1, characterized in that, said G ₂ is -C(=O)OR;

   And/or, said G ₄ is -C(=O)OR';

   And/or, said R ₂ is methyl, and said R ₃ is hydrogen.
4. The ionizable lipid compound according to claim 1, wherein the ionizable lipid compound is one or more of the following compounds:

   

   
5. A delivery carrier, characterized in that the delivery carrier includes one of the ionizable lipid compounds represented by the general formula (I) and the general formula (II) according to any one of claims 1 to 5 one or more species.
6. The delivery carrier according to claim 6, characterized in that, the delivery carrier also includes auxiliary molecules, and the molar ratio of the ionizable lipid compound and the auxiliary molecules is (0.1-1 ): (0.1~1).
7. The delivery carrier according to claim 7, characterized in that, the auxiliary molecules include artificially synthesized or naturally derived auxiliary lipids or lipid molecules, any species of animal sources, and any types of cells or vesicles , one or more of polypeptide molecules, polymer molecules, carbohydrate molecules or inorganic substances.
8. The delivery carrier according to claim 8, wherein the auxiliary molecules include cholesterol, calcipotriol, stigmasterol, lupeol, β-sitosterol, betulin, ursolic acid, oleanol, AHA, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, 1-stearyl-2-oleoyl lecithin, dioleoylphosphatidylethanolamine, (1,2-dioleoxypropyl base) trimethylammonium chloride, didecyldimethylammonium bromide, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, dipalmitoylphosphatidylethanolamine-methoxy One or more of polyethylene glycol 5000, distearoylphosphatidylethanolamine-polyethylene glycol 2000, activated carbon, silicon dioxide, and calcium phosphate.
9. The delivery carrier according to claim 7, characterized in that, the ionizable lipid compound and/or the auxiliary molecule is modified with targeting substances, and the targeting substances include folic acid, single-chain One or more of antibodies or targeting polypeptides.
10. The delivery carrier according to claim 6, characterized in that, the delivery carrier is a nano-lipid particle, and the particle size of the nano-lipid particle is 50nm-200nm; and/or, the nanoparticle preparation polydispersity index≤0.4. 12. The delivery carrier according to claim 11, characterized in that, the delivery carrier can deliver nucleic acid molecules, and the nucleic acid molecules include but not limited to plasmid DNA (pDNA), siRNA, One or more of ASO or mRNA; and/or, the mass ratio of the nucleic acid molecule to the delivery carrier is 1:(5-50).
11. The delivery carrier according to claim 9, characterized in that, the delivery carrier and the nucleic acid molecules form a nucleic acid pharmaceutical composition, and the nucleic acid pharmaceutical composition also includes pharmaceutically acceptable additives, and the additives include One or more of excipients, stabilizers or diluents.
12. The delivery carrier according to claim 13, characterized in that, the added amount of the additive is 1%-20% of the total mass of the pharmaceutical composition.
13. The delivery carrier according to claim 6 or 13, characterized in that, the delivery carrier or the nucleic acid pharmaceutical composition is lyophilized powder or injection, which can be injected through muscle, subcutaneous, endothelial, intratumoral, microneedle, Or local administration by infusion, or administration by intravenous injection.
14. A method for delivering nucleic acid molecules in vivo, characterized in that the delivery vector according to any one of claims 6 to 15 is used to deliver the nucleic acid molecules into the body of a subject.
15. The method for delivering nucleic acid molecules in vivo according to claim 16, wherein the subject is a mammal.
16. The method for delivering nucleic acid molecules in vivo according to claim 16 or 17, wherein the subject is a human.

Images